Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways

Daeshiho-tang (DSHT), a traditional herbal formula with diverse pharmacological effects, has shown promise in medicine owing to its anti-hypertensive, anti-diabetic, and anti-inflammatory properties. However, the precise molecular mechanism underlying these effects remains unclear. Thus, we investigated the effect of DSHT on inflammatory response and oxidative stress to understand its molecular mechanism using lipopolysaccharide (LPS)-induced macrophage (RAW 264.7) cells. DSHT decreased the contents of nitric oxide (NO) and prostaglandin E2 (PGE2) through downregulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions. DSHT suppressed the LPS-induced TLR4 as well as MyD88, subsequently suppressing the NF-κB activation and the phosphorylation of MAPK (p38, ERK, and JNK). Radical scavenging activity results revealed a dose-dependent response of DSHT with diminished ABTS activity, a hallmark of oxidative stress potential. Furthermore, DSHT enhanced Nrf2 and HO-1 expression in response to LPS. Collectively, our findings indicated that DSHT exert anti-inflammatory effect and regulating oxidative stress by modulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways, consequently can provide potential therapeutic strategy for the prevention and treatment of inflammation and oxidative stress-related diseases.

www.nature.com/scientificreports/ in turn induced oxidative stress, consequently, contributing to the development of inflammation and abnormal inflammatory cytokine production 4,19 .Further, increased ROS level in LPS-stimulated macrophages not only sustain inflammation but also drive excessive production of inflammatory cytokines 4,19 .

DSHT induces cell proliferation against macrophages
We first investigated the impact of DSHT on cell proliferation using RAW 264.7 cells.As illustrated in Fig. 1A, DSHT promoted a concentration-dependent elevation in cell proliferation, with a notable increase of 107.8-140.8%.To evaluate the effects of DSHT on morphology, macrophages were exposed to varying concentrations of DSHT and/or LPS (Fig. 1B).In the absence of LPS, DSHT-treated macrophages exhibited no marked morphological alterations, maintaining their round shape similar to that in the normal state.Conversely, LPStreated macrophages underwent a morphological transition, adopting a polygonal spindle-shaped pseudopodia, a hallmark of macrophage activation, and treatment with DSHT had no effect on cell morphology.

DSHT inhibits LPS-induced NF-κB pathway by regulating its translocation
To clarify the correlation between DSHT's anti-inflammatory activity and inflammatory signaling pathways, we assessed DSHT activity on IκBα and NF-κB and its underlying mechanism.As shown in Fig. 4, LPS promotes the nuclear accumulation of NF-κB concomitant with decreased cytosolic IκBα and NF-κB, which implies NF-κB activation.In terms of translocated NF-κB, DSHT exhibited dose-dependent reduction of 15.2% and 42.1% (p < 0.05) at 100 and 400 μg/mL, respectively, with a significant effect observed at 400 μg/mL.

DSHT blocks the transduction of LPS-mediated TLR4 pathway
To verify whether the impact of DSHT on the regulation of NF-κB and MAPK in terms of its anti-inflammatory effects was related to the TLR4/MyD88 pathways, we assessed the effect of DSHT on TLR4 and MyD88 expression.As illustrated in Fig. 6, the TLR4 and MyD88 expressions were increased by LPS, but there was no significant induction observed in MyD88 expression.Meanwhile, TLR4 expression revealed a tendency to be significant following LPS stimulation.Akin to the MAPK results, both the TLR4 and MyD88 expressions decreased by DSHT in concentration-dependent manner and were reduced to 52.9% (p < 0.05) and 60.3% (p < 0.01), respectively, at 400 μg/mL, but there was no significant reduction in MyD88 at 100 μg/mL.

DSHT regulates ABTS radical scavenging activity
To elucidate whether the impact of DSHT's activity is associated with the oxidative stress, we evaluated 2,2'-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS) radical scavenging activity (Fig. 7).Trolox, which served as a free radical scavenger, exhibited potent ABTS radical scavenging activity and significantly increased by up to 80%.Similarly, DSHT showed potent scavenging activity against ABTS radical and remarkably induced

Discussion
In this study, we used an LPS-stimulated macrophages to ascertain whether DSHT can modulate the inflammatory response and oxidative stress.The results indicated that DSHT has anti-inflammatory impact induced by LPS through regulating inflammatory signaling and oxidative stress, such as TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways.Excessive release of inflammatory cytokine from activated macrophages make them a potential target for regulating inflammation and inflammatory diseases 31 .NO, synthesized from L -arginine through the iNOS, holds a pivotal role in physiological conditions.However, LPS-stimulated macrophages overexpress iNOS, causing excessive NO production.This not only contributes to the development of inflammatory disease but also induced oxidative stress 32,33 .Meanwhile, COX-2, a catalytic enzyme, converts arachidonic acid to generate PGE 2 , and excessive PGE 2 is induced by COX-2 leads to various inflammatory responses 16,33,34 .Suppression of NO and PGE 2 has emerged as a promising therapeutic strategy against inflammation-related disorders.Our findings demonstrated that DSHT dose-dependently inhibited LPS-induced NO and PGE 2 secretion in macrophages, which was related with the inhibition of iNOS and COX-2.
Among the macrophage activation-associated intercellular signaling pathways, transcription factor NF-κB is crucial for regulating the inflammatory response.In a physiological setting, it remains inactive in the cytoplasm while complexed with IκBα 35,36 , however, upon exposure to irritant such as LPS, NF-κB translocate into the nucleus, triggering the transcriptional activation of inflammation-related genes.Another key player, MAPK, a serine/threonine protein kinase, includes p38, ERK, and JNK.These kinases influence various cellular processes including proliferation, stress response, inflammation, and apoptosis.Furthermore, MAPK orchestrated the regulation of pro-inflammatory mediators and expression of cytokines 37 .The cell surface receptor, TLR4, induced by LPS, is capable of recruiting MyD88 to activate NF-κB and MAPKs.To understand the regulatory mechanism underlying the effect of DSHT on the LPS-stimulated inflammatory response, we assessed the expression level of TLR4/MyD88, NF-κB, and MAPK pathways.Our results revealed that DSHT reduced LPS-triggered TLR4 and MyD88 expression.Furthermore, DSHT exhibited the ability to modulate the phosphorylation of ERK and JNK, suggesting its role in the inhibition of MAPK signaling pathway on LPS-triggered macrophage activation.Additionally, in the nucleus, DSHT significantly reduces the expression level of NF-κB in LPS-stimulated macrophages  Oxidative stress, an imbalance between oxidants and antioxidants, arises from excessive production of ROS.While ROS are essential molecules for biological processes, their overproduction contributes to the production of inflammatory cytokines and progression of inflammatory-related disease 4,18 .Therefore, the regulation of oxidative stress is important to maintain a balance in ROS generation.ABTS, commonly used to measure radical scavenging activity, reacts with potent antioxidants.DSHT induced a dose-dependent ABTS radical scavenging activity, demonstrating its antioxidant effects.HO-1, recognized for its pivotal role in the antioxidant pathway 38 , Also exerts control over the generation of anti-inflammatory cytokines and mediators 39,40 .Nrf2, major regulator of redox homeostasis, is also involved in oxidative stress and inflammatory response through the induction of detoxifying enzyme including HO-1.This study revealed that DSHT slightly induced the expression of Nrf2 and HO-1 in the presence of LPS.Furthermore, co-administration of LPS and DSHT significantly increased their expression level compared to LPS alone.These observations collectively demonstrate that the ABTS radical scavenging effect of DSHT was associated with the enhancement of HO-1 through Nrf2 activation in the presence of LPS, thus alleviating oxidative stress.

Conclusion
Our study demonstrated that DSHT regulated LPS-induced inflammatory response and mitigated oxidative stress, a modulation of intricately linked to the suppression of TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 signaling transductions.These findings collectively underscored the potential pharmacological benefits of DSHT for the prevention and treatment of disorders related to inflammation and oxidative stress.